Glucose uptake and metabolism are critical to life. It is therefore not surprising that modulation of these processes affects cell death pathways. Over the last year or two, however, it has become apparent that the role of glucose metabolism in regulating programmed cell death is even more critical than initially suspected. We and others have found that glucose transporter expression and its effects on glucose uptake and metabolism often determine whether cell death will occur and serve as important mediators of signaling pathways that directly regulate cell death pathways. Our initial studies focused on the interaction of glucose transport and of hypoxia in vascular smooth muscle cell apoptosis. We found that over expression of the facilitative glucose transporter, GLUT 1, protected vascular smooth muscle cells and cardiac myocytes from hypoxia-induced apoptosis and determined that such protection required glycolysis. We also have shown that glucose transporter expression and glucose metabolism prevent activation of the mitochondrial cell death pathway. We have also determined that GLUT1 over expression and glucose metabolism directly affect expression or activity of important upstream mediators of the mitochondrial cell death pathway, including Hif-1-alpha,p53, INK, Akt and GSK-3ft. Because apoptosis is an important mediator of vascular change under both normal and pathological conditions, we have continued to focus on vascular smooth muscle cells as our model system. Our major hypothesis for continuation of these studies is that glucose transport and metabolism, especially as controlled by expression of facilitative glucose transporters, directly protects VSMCs from apoptosis induced by multiple cell stresses and does so through specific effects on multiple upstream regulators of the mitochondrial apoptotic pathway. Certain signal transduction pathways including the Hif-1a/p53, Akt/GSK3 and iNK pathways have been implicated in the control of the mitochondrial cell death pathway by work in our and other laboratories. In addition, our preliminary data suggest that glucose transport and metabolism may specifically modulate apoptosis by the effects on these pathways. Therefore, we propose the following specific aims: 1. Determine the effects of Hif- I a/p53, Akt/GSK3, and JNK expression and activation on hypoxia-induced apoptosis in VSMCs. 2. Determine the effects of glucose uptake, glycolysis, and glucose transporter expression on Hif-1alpha/p53 Akt/GSK3, and JNK expression and activation in VSMCs. Establish whether these effects are responsible for glucose and GLUTI-induced protection from apoptosis. 3. Determine effects of glucose uptake, glycolysis, and glucose transporter expression on aspects of mitochondrial metabolism, which regulate apoptosis. Establish whether these effects are responsible for glucose and GLUTI. To test our hypothesis we will utilize molecular and cell biological techniques to specifically modulate expression of various putative upstream signals in cultured VSMCs to determine which are most critical in the initiation of hypoxia-induced apoptosis. We will then determine in what manner the most critical of these pathways are affected by glucose transporter expression and by modulation of other glucose metabolic enzymes. We will also determine the mitochondrial metabolic effects that are most critical to both hypoxia-induced apoptosis and glucose protection in VSMCs.